Sex-dependent phosphorylation of Argonaute 2 reduces the mitochondrial translocation of miR-181c and induces cardioprotection in females

Obesity-induced cardiac dysfunction is growing at an alarming rate, showing a dramatic increase in global prevalence. Mitochondrial translocation of miR-181c in cardiomyocytes results in excessive reactive oxygen species (ROS) production during obesity. ROS causes Sp1, a transcription factor for MICU1, to be degraded via post-translational modification. The subsequent decrease in MICU1 expression causes mitochondrial Ca²⁺ accumulation, ultimately leading to a propensity for heart failure. Herein, we hypothesized that phosphorylation of Argonaute 2 (AGO2) at Ser 387 (in human) or Ser 388 (in mouse) inhibits the translocation of miR-181c into the mitochondria by increasing the cytoplasmic stability of the RNA-induced silencing complex (RISC). Initially, estrogen offers cardioprotection in pre-menopausal females against the consequences of mitochondrial miR-181c upregulation by driving the phosphorylation of AGO2. Neonatal mouse ventricular myocytes (NMVM) treated with insulin showed an increase in pAGO2 levels and a decrease in mitochondrial miR-181c expression by increasing the binding affinity of AGO2-GW182 in the RISC. Thus, insulin treatment prevented excessive ROS production and mitochondrial Ca²⁺ accumulation. In human cardiomyocytes, we overexpressed miR-181c to mimic pathological conditions, such as obesity/diabetes. Treatment with estradiol (E₂) for 48 h significantly lowered miR-181c entry into the mitochondria through increased pAGO2 levels. E₂ treatment also normalized Sp1 degradation and MICU1 transcription that normally occurs in response to miR-181c overexpression. We then investigated these findings using an in vivo model, with age-matched male, female and ovariectomized (OVX) female mice. Consistent with the E₂ treatment, we show that female hearts express higher levels of pAGO2 and thus, exhibit higher association of AGO2-GW182 in cytoplasmic RISC. This results in lower expression of mitochondrial miR-181c in female hearts compared to male or OVX groups. Further, female hearts had fewer consequences of mitochondrial miR-181c expression, such as lower Sp1 degradation and significantly decreased MICU1 transcriptional regulation. Taken together, this study highlights a potential therapeutic target for conditions such as obesity and diabetes, where miR-181c is upregulated. New and noteworthy: In this study, we show that the phosphorylation of Argonaute 2 (AGO2) stabilizes the RNA-induced silencing complex in the cytoplasm, preventing miR-181c entry into the mitochondria. Furthermore, we demonstrate that treatment with estradiol can inhibit the translocation of miR-181c into the mitochondria by phosphorylating AGO2. This ultimately eliminates the downstream consequences of miR-181c overexpression by mitigating excessive reactive oxygen species production and calcium entry into the mitochondria.